We previously showed using microarray profiling biotechnology that stepwise transformation and metastatic progression of SCC in a murine model results in the expression of gene programs related to the signal transcription factor NF-kappaB. Inhibition of NF-kappaB modulated over half the up or down-regulated genes differentially expressed, and attenuated the malignant phenotype, indicating it may be a critical target for prevention or therapy of head and neck cancer. Gene expression profiling and bioinformatic analysis of the promoters of gene clusters differentially expressed in human HNSCC provided evidence for increased prevalence of binding motifs for NF-kappaB as well as other signal transcription factors, such as p53, AP-1, STAT3 and EGR-1 (Yan et al, Genome Biology, 2007). NF-kappaB, p53, AP-1, STAT3 and EGR-1 activation has previously been associated with pathogenesis and therapeutic resistance, and the subsets expressing wt or mt 53 have been reported to differ in response to chemotherapy. These observations suggested the hypothesis that key alterations in a network of signal transcription factors can interact in determining gene expression and development of HNSCC of differing malignant potential and sensitivity or resistance to therapy. Next, we took a systems biology approach to define NF-kappaB regulons, interacting signal pathways and networks in the malignant phenotype of head and neck cancer cell lines differing in p53 status(Yan, Genome Biology, 2008). Unique gene signatures expressed by human HNSCC lines were identified by cDNA microarray, principal components, and cluster analyses and validated by quantitative reverse transcription-PCR (RT-PCR) and in situ hybridization. An inverse relationship between activation of NF-kB, p53 mutation, and inactivation of TGFbeta pathway and target genes suggested the hypothesis that activation of NF-kB may be linked to alteration of p53 and TGFbeta signaling. We discovered that mt p53 represses TGFbeta receptor II expression, attenuating TGF beta signaling, enhancing inflammatory cytokine responsiveness and activation of NF-kB in HNSCC (Cohen et al, Cancer Res 2009). Another relationship identified was between NF-kB member c-REL and p53 members p53, p63 and p73. This led to demonstration that cytokine TNF induced cREL interacts with p63, displacing p73 from growth arrest and apoptotic genes and the nucleus of HNSCC (Lu et al, manuscript submitted). siRNA knockdown of p63 revealed that it down regulates p53 regulated tumor suppressor genes and upregulated numerous NF-kB related genes, through interaction with c-REL. Novel p63, cREL as well as classical p53 and NF-kB sites were defined in these genes and validated by ChIP assay (Yang et al, manuscript in preparation). ChIP sequencing of global gene expression regulated by cREL, p63 and p73 in HNSCC is underway in collaboration with Laboratory of Genetics, NCI. In collaboration with the NCI and Department of Pathology, new proteomic technology, using protein microarrays, was used to analyze EGFR, NF-kB, MAPK, AKT and STAT3 signal phosphoprotein inhibition, and markers of apoptosis in protein extracts from HNSCC lines and specimens from patients in a clinical trial with small molecule inhibitor gefitinib, taxol and radiation.